Decades of research have provided numerous insights as to how Cofilin interacts with and alters actin. However, these actions have complex outputs in the cell, including promoting both assembly and breakdown of actin networks. Thus the same activity can lead to opposite outcomes. For example, the local upregulation of Cofilin has been shown to be both associated with attractive and repulsive growth cone guidance. It is likely that that these seemingly conflicting results are mediated by where, when, and how much Cofilin is being activated. Several studies have shown that localization and timing of Cofilin activation is critical in determining which downstream behaviors it invokes. However, the tools to directly test these ideas have been limited. We hypothesize that a methodology which allows us to instantly inactivate Cofilin with subcellular precision will lead to the discovery of new mechanistic information of how Cofilin functions to regulate actin networks and to control growth cone motility. To determine the spatiotemporal role of Cofilin activity during growth cone migration, we propose the following Specific Aims: (1) To develop a methodology for local and instantaneous inactivation of Cofilin; (2) to determine the effects of instantaneous inactivation of Cofilin on actin distribution and dynamics in growth cones; and (3) to determine the effects of local inactivation of Cofilin on growth cone motility. Using a technique called Chromophore Assisted Laser Inactivation (CALI), we will show that we are able to instantly inactivate Cofilin with subcellular precision. We will develop this methodology so that it will be a generally applicable and useful tool for other labs who want to determine the functional consequences of local Cofilin inactivation. By combining CALI with high resolution live cell microscopy, we will monitor actin network changes in real time after instantaneous Cofilin depletion. Finally, we will use CALI to determine how local inactivation of Cofilin effects growth cone migration and guidance. Defects in axon guidance are associated with developmental disorders and nerve regeneration failure. Understanding the fundamental biological processes that underlie axon growth will allow for the design of better, more effective disease treatments. Cofilin has been implicated in growth cone motility, but its complex functional role has yet to be fully elucidated. In this proposal, we will uncover new mechanistic information about how Cofilin functions to regulate axon growth so that its role in guidance related disorders can be better understood.